Patient Derived Xenograft & Patient Derived OrganoidsPatient Derived Xenograft & Patient Derived Organoids

Question 1

Modeling tumour biology can be completed using which 2 in vitro pre-clinical laboratory cancer models?

Answer 1

– 2D cancer cell line cultures

– Patient derived organoids “PDO”

Question 2

How are 2D cancer cell line cultures generated?

Answer 2

2D immortalized clonal cell lines are traditionally grown in a monolayer

• The oldest human cell line was derived from cervical cancer cells (HeLa cells) in 1951
• NCI-60 Cell Line screen (1990)- 60 cell lines represented various cancers, and screened various drugs to inform new drug discovery in multiple cancer types

Question 3

Why use 2D in vitro models?

Answer 3

• Banks of cell lines allow access to many genotypic
backgrounds of tumours from different tissue types

• Genomic, epigenetic and transcriptomic characterisation
• Suitable for High throughput drug screening
• Have been shown to faithfully recapitulate a number of specific aspects of cancer biology
• Screening of cell lines have been used to identify therapeutic biomarkers

Question 4

What are the limitations of 2D in vitro models?

Answer 4

Unable to fully capture tumour heterogeneity to directly predict responses in the clinic

Monolayer culture:
• Limited cell-cell contact.
• Limited complex interactions with adjacent cells • 3D complexity is lost

Change in morphology:
• Lack of relevant environmental cues (matrix)

Question 5

What is an ideal in vitro model?

Answer 5

Suitable for culture

• Ability to proliferate and expand
• Stability in culture

Relevance to patients

• Presence of all cell types within a tumour
• Genotypic characteristics
• Recapitulate architecture of original tissueAnalysis
• Medium/ high throughput
• Reproducibility
• Advanced technological screening

Question 6

What is an organoid?

Answer 6

A structure resembling the organ from which it is derived

• Cultured in 3D

• Organ-specific cell
types

• Self organizing

• Capable of continuous
expansion in vitro

Question 7

How are 3D organoid models developed?

Answer 7

E.g Isolating Intestinal crypt

+ growth factors
+ supporting matrix

Sustainable growth by mimicking the intestinal environment

• hydrogel to maintain 3D integrity
• Growth factor supplements to support stem cells

Question 8

How are tumour organoid models developed?

Answer 8

Growth conditions further adapted to culture material from patient tumours

Tissue from patient biopsies

Dissection, digestion, trituration

Isolated cell fragments from tissue

3D culture in matrix
Optimised media conditions

Question 9

Organoids have been derived from multiple tissue types

True or false

Answer 9

True

Question 10

What are the advantages of PDOs as a pre-clinical tool?

Answer 10

Morphology
• Similar histology to
matching patient tumours
• Retain similar cell signaling to native tissue due to 3D architecture

Genetic representation of tumours
• Have the capacity to maintain heterogeneity of tumours as analyzed by DNA sequencing

• Protein expression well retained across different tumour organoid type versus originating material
• Genetic diversity of cancer is represented

Question 11

What are future opportunities for PDOs to model patient responses in the clinic?

Answer 11

PDO generation from an individual patient

Compare against sequence of patient tumour

Drug screen in patient-derived model to identify effective treatments

Question 12

What is an example of a Co-clinical trial with PDOs?

Answer 12

Able to derive from cells from metastatic colon cancer patients pre and post treatment with chemotherapy Paclitaxel, able to establish an organoid that was responsive to Paclitaxel, therapy, whereas when attained patient tumour from the resistant metastatic biopsy, found that the organoid was resistant.

Question 13

What are disadvantages of PDOs for modeling patient tumours?

Answer 13

• Protocols currently support models that are epithelial

– Lack other cell types present in the tumour micro- environment

– Not suitable for immunotherapy testing

– Co-culturing methods possible but require further development

• Derivation of model can vary depending on source tissue and quality of initial sampling
• Overgrowth with normal epithelial tissue in culture can be an issue with some models e.g. prostate PDOs
• Need rigorous testing before applicable for pre-clinical drug discovery

Question 14

What are the three in vivo models in cancer?

Answer 14

• Genetically engineered mouse (GEM) models
• In vivo cell line models
• Patient Derived Xenograft (PDX) models

Question 15

How are genetically engineered mouse (GEM) models formed?

Answer 15

Generated through introduction of genetic mutations associated with cancer of interest

Involves:

• Embryo editing
• Gain of function (oncogenes)/ loss of function (tumour suppressors)
• Knock-in/ Knock-out
• Ubiquitous or site specific (tissue specific promoter)
• Constitutively or conditionally expressed (inducible)
• Result in development of spontaneous tumours

Question 16

What are the pros of using genetically engineered mouse models?

Answer 16

• Tumours occur in the relevant in situ environment
• Tumours are heterogeneous and polyclonal
• Intact immune system – allows modelling of tumour microenvironment

Question 17

What are the cons of using genetically engineered mouse models?

Answer 17

• Models slow to develop – can take several years

Question 18

What are two types of In vivo cell line models?

Answer 18

Syngeneic model – mouse cell line in immunocompetent mice

Xenograft model – human cell line in immunocompromised mice - - Genetically modified immune system so that human cells are not rejected

Question 19

What are three Implantation Sites in an in vivo cell line models?

Answer 19

• Ectopic – subcutaneous injection
• Orthotopic – site specific e.g. specific organ
• Metastatic model – injection into bloodstream

Question 20

What are the pros of in vivo cell line models?

Answer 20

• Well characterized cell lines
• Can screen drug candidates in vitro before moving in vivo
• Easy to obtain large numbers of cells for in vivo experiments

Question 21

What are the cons of in vivo cell line models?

Answer 21

• Do not capture the heterogeneity of human tumours
• Long term growth in vitro can result in aberrant selection pressures
• Often derived from highly aggressive metastatic tumours – less useful for modelling events in evolution of primary tumour

Question 22

What are in vivo cell line models?

Answer 22

Cell lines originally derived from patient tumours and immortalised to continuously grow in 2d cell cultures. Can then be transplanted into mouse hosts.

Question 23

What are Patient-Derived Xenograft (PDX) models

Answer 23

Implantation of patient tumour into immunocompromised mice

Serially passaged through further mice to generate banks of material

Question 24

PDX models can be established using tumours from which sample types?

Answer 24

• Primary tumour/ Metastatic site
• Surgical sample/ Biopsy/ Pleural effusion
• Single Cells/ Tumour fragments

Question 25

As cell models, where can PDX samples be implanted?

Answer 25

Ectopic – subcutaneous injection

Orthotopic – site specific e.g. specific organ

Question 26

What are was found when metastatic Colorectal Cancer PDXs: Response to cetuximab (EGFR Ab) treatment was investigated?

Answer 26

Metastatic Colorectal Cancer PDXs: Response to cetuximab (EGFR Ab) treatment:

Using PDX models found that a large proportion of KRAS WT PDXs that did not respond to cetuximab had HER2 amplification

Dual targeting of HER2 and EGFR induced tumor regression in PDXs from KRAS WT, HER2 amplified, cetuximab resistant patients

Drugs: Pertuzumab – mAB, disrupts HER2 dimerization with EGFR

Lapatinib – small molecule inhibitor or HER2 + EGFR

Question 27

PDX models can be used as avatars to predict patient responses to treatment

What is an example?

Answer 27

Irinotecan, cetuximab and bevacizumab resulted in marked decrease in tumour volume in PDX

Treatment of patient with irinotecan, cetuximab and bevacizumab resulted in marked decrease in tumour volume

Question 28

What are limitations of PDX models in cancer research?

Answer 28

• Rate of engraftment of PDX models can vary
• Mice are immunocompromised = loss of interaction between tumour and immunological component
• Expensive and labour intensive compared to cell line models
• Rigorous pre-clinical testing involves high numbers of mice = limits of large-scale testing.
• [Gao et al (2015) have suggested this could be circumvented using a ‘1 PDX per patient model per treatment’ approach]

Question 29

Tumour biology can be modelled using what 3 pre-clinical laboratory cancer models?

Answer 29

– 2D cancer cell line cultures
– Patient derived organoids “PDO”
– Patient derived xenografts “PDX”